The present invention relates to parallel methods for isolating nucleic acids from cells. In certain embodiments, the invention is focused on the isolation of self-replicating extrachromosomal DNA such as plasmid DNA. Commercially available formats for high-throughput purification of plasmid DNA include plates, spin columns and magnetic beads. Although attempts have been made to automate these technologies, they still employ a manual step of isolating cells from the medium in which they are grown, usually by centrifugation.
Conventional wisdom teaches that it is necessary to remove the growth medium from the cells before isolation of nucleic acid. In most plasmid preparation methods, the culture is centrifuged after growth and the spent medium supernatant is removed (see for example U.S. Pat. Nos. 6,277,648 and 6,297,371). This centrifugation step is not automated and requires human intervention, time and expense. Therefore, there exists a need for a fully automated method for preparation of plasmid DNA that does not require separation of cells from the growth medium. That is, there exists a need to recover plasmid DNA directly from a culture without the removal of the medium, associated byproducts, cell debris and other materials. The present invention fills this need and provides a fully automated method for nucleic acid isolation without centrifugation.
After the medium is removed, the next step in the isolation of nucleic acids is cell lysis and removal of cell debris, which is most often accomplished with a second centrifugation step. Conventional wisdom additionally teaches that this second centrifugation step is required for successful nucleic acid isolation. For example, U.S. Pat. No. 5,990,301, entitled “Process for the Separation and Purification of Nucleic Acids from Biological Sources” states that the problem of separating lysed cells from their contents is most effectively solved by centrifugation (column 6, lines 33-46). U.S. Pat. No. 6,368,800 entitled “Kits for Isolating Biological Target Materials using Silica Magnetic Particles”, states that cellular debris is likely to interfere with the adhesion of nucleic acids to silica magnetic particles (column 10, lines 49-59). An automated method was developed to bypass this second centrifugation step which is the subject of international Application No. PCT/US11/30232, which is incorporated by reference herein.
In other embodiments, the invention can be used to isolation genomic DNA. Genomic DNA can be extracted and purified from samples such as mouse tails, blood, saliva, tissues from biopsies, cerebrospinal fluid, animal tissues, plant tissue and whole organisms such as fruit flies, worms and embryos. Sample preparation can be performed in a completely automated fashion in 96-well format.